Anemoside B4 inhibits enterovirus 71 propagation in mice through upregulating 14-3-3 expression and type I interferon responses.

Enterovirus 71 (EV71) is the major pathogens of human hand, foot, and mouth disease (HFMD). EV71 efficiently escapes innate immunity responses of the host to cause infection. At present, no effective antiviral drugs for EV71 are available. Anemoside B4 (B4) is a natural saponin isolated from the roots of Pulsatilla chinensis (Bunge) Regel. P. chinensis extracts that shows a wide variety of biological activities. In this study, we investigated the antiviral activities of B4 against EV71 both in cell culture and in suckling mice. We showed that B4 (12.5-200 µM) dose dependently increased the viability of EV71-infected RD cells with an IC50 value of 24.95 ± 0.05 µM against EV71. The antiviral activity of B4 was associated with enhanced interferon (IFN)-ß response, since knockdown of IFN-ß abolished its antiviral activity. We also confirmed that the enhanced IFN response was mediated via activation of retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) pathway, and it was executed by upregulation of 14-3-3 protein, which disrupted the interaction between yes-associated protein (YAP) and interferon regulatory factor 3 (IRF3). By using amino acids in cell culture (SILAC)-based proteomics profiling, we identified the Hippo pathway as the top-ranking functional cluster in B4-treated EV71-infected cells. In vivo experiments were conducted in suckling mice (2-day-old) infected with EV71 and subsequently B4 (200 mg · kg-1 · d-1, i.p.) was administered for 16 days. We showed that B4 administration effectively suppressed EV71 replication and improved muscle inflammation and limb activity. Meanwhile, B4 administration regulated the expressions of HFMD biomarkers IL-10 and IFN-γ, attenuating complications of EV71 infection. Collectively, our results suggest that B4 could enhance the antiviral effect of IFN-ß by orchestrating Hippo and RLRs pathway, and B4 would be a potential lead compound for developing an anti-EV71 drug.

Hederacolchiside C inhibits Enterovirus 71 propagation through activating innate immunity.

Enterovirus 71 (EV71), a newly emerging life-threatening pathogen induces hand-foot-mouth disease (HFMD), no effective vaccines or specific anti-viral treatments are currently available. In this study, the activity of hederacolchiside C (HSC) against EV71 was investigated, and the antiviral mechanism was explored. HSC displayed apparent antiviral activity in EV71-infected cells probably through activating the host innate immunity. Comparing with EV71-infected group at 24 hpi, the group pretreated with HSC dramatically increased the expression of MAVS, p-IRF3, IRF3 and IFN-ß, the innate immune effectors related to innate immunity. In addition, HSC displayed stronger antiviral activity in EV71-infected suckling mice in comparison with Ribavirin, a broad-spectrum antiviral drug. The results suggest that HSC could have potential as a pharmaceutical drug for HFMD.

[Application of flow cytometry to detect PP65 antigenemia for diagnosis and monitoring of human cytomegalovirus infection].

OBJECTIVES: To evaluate the clinical significance of flow cytometry (FCM) to detect the cytomegalovirus (CMV) PP65 antigen in patients with CMV infection. METHODS: Samples from 35 patients without CMV infection were used as negative control. The definite diagnosis of CMV infection was based on the national criteria for CMV infection. All 136 patients with CMV infection were examined with the FCM to detect CMV PP65 antigen, real-time fluorescence quantitative-polymerase chain reaction assay (RFQ-PCR) to detect CMV-DNA and ELISA to measure the serum level of IgM antibody against CMV. The results of these 3 assays in 2 groups (isolated organ involvement and disseminated diseases) were compared and the significance of PP65 antigenemia was evaluated. A short-term follow-up was undertaken in 18 patients. RESULTS: The percentages of PP65 positivity in blood mononuclear cells (MNC) and polymorphic nuclear leukocyte (PMNL) from 35 negative control patients were 0.21% +/- 0.09% with a range of 0 - 0.41% and 0.24% +/- 0.10% with a range of 0.12% - 0.48%, respectively, which were not significantly different (t = 0.425, P > 0.05). The 95(th) percentiles (P(95)) of PP65 in MNC and PMNL were 0.39% and 0.45%, respectively, so a cutoff value of >/= 0.50% was set. Of the 136 patients with CMV infection, 118 samples from 118 patients were positive for PP65 antigenemia with a positive rate of 86.8%, which was not statistically different from that (90.4%, chi(2) = 0.91, P > 0.05) of CMV-DNA detected by RFQ-PCR assay but it was significantly higher than that (45.6%, chi(2) = 51.50, P < 0.005) of the detection by IgM measurement. PP65 detection was correlated with urine CMV DNA amplification (chi(2) = 63.78, P < 0.01) while the different detection rates between the two assays were not statistically significant (chi(m)(2) = 1.78,P > 0.05). PP65 detection was not correlated with serum IgM measurement while the detection rates between the two were significantly different (chi(m)(2) = 52.92,P < 0.01). No significant difference was found between the detection rates of CMV infection in MNC (45/53, 84.9%) and PMNL (43/53, 81.1%) (chi(m)(2) = 0.25, P > 0.05). Higher PP65 antigenemia level was correlated with systemic CMV infection, while lower level of PP65 was either in the patients with isolated organ involvement by CMV (chi(2) = 38.51, P < 0.005) or less severe in patient's situation. PP65 antigenemia of CMV infection returned to lower level or negative in recovery stage and increased when condition of patients deteriorated. CONCLUSIONS: PP65 antigenemia detection by FCM is effective in the diagnosis of the active CMV infection. Quantitative monitoring of PP65 antigenemia is useful in the evaluation of patients with CMV infection.